The use of microfabricated and microfluidic structures in biomedical research has been rapidly expanding in recent years, but the number of commercial products and the applications they address remain limited. Most research applications of these devices require customization, and new applications typically require several design iterations for troubleshooting. In an effort to bring the necessary technical capabilities and knowledge to do so into biomedical laboratories, we have developed a basic in-house microfabrication facility accessible to researchers across the intramural program. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. We are able to reliably pattern single- and multi-layer template features with lateral dimensions of less than 2 microns, and with heights ranging from a few microns to a few hundred microns. We have developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, including techniques for making and manipulating thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. We can also perform surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, and have developed techniques for connecting devices to flow-control instruments such as syringe pumps and pressure controllers. In addition, we have the ability to deposit and pattern metal layers, as well as a heated hydraulic press for hot-embossing thermoplastics, including PMMA, polycarbonate, and COC. We have also used a programmable razor cutter and pressure-sensitive adhesive to directly make thin film structures with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This is a low-cost and convenient method for several applications, including the ready fabrication of flow cells with two glass walls, or for fluidic confinement over already-functionalized surfaces. Finally, we continue to work on finite element modeling of transport in microfabricated structures, developing models for oxygen delivery in a bioreactor with micropillars and for chemokine concentration in a microfluidic hydrogel device. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NEI, NIAID, NHLBI, NCI, NICHD, NINDS, NIDCD, NIDDK, and NIBIB. 1) A collaborative effort with LSB, NIAID, to study chemotaxis of primary immune cells in 3-D collagen matrices, using a microfluidic agarose device to generate reproducible time-varying spatial gradients on a platform compatible with high-resolution fluorescence and two-photon imaging. Finite element models of the original device have been used in combination with quantitative image analysis to assist in characterization and modeling of the 3-D device for different temporal inputs. 2) The fabrication and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, light-activated microdissection. This work was begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, and more recent efforts with NICHD, NCI, and CIT have focused on refining the instrumentation and microdissection protocols for a variety of targets and stain localizations, from whole-cell and region-of-interest based captures, to subcellular targets, such as axon terminals in brain slices (in collaboration with NIDA). 3) The design, fabrication, modeling, and use of an oxygen-transmissive membrane, patterned with a micropillar array to deliver oxygen to three-dimensional cell culture volumes with in vivo-like spatial distribution, in collaboration with LCB, CCR, NCI and SPIS, CIT. Because the pillar spacing is approximately equal to typical intercapillary distances (200 microns), cells in a Matrigel layer surrounding the pillars can be maintained under hypoxic conditions in an extended 3D volume. We recently implemented a higher throughput system that incorporates the membranes into a multiwell plate format, and have begun using the system to study a variety of cellular models. 4) The development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. We have successfully used these removable devices for deposition of linear and exponential CSPG gradients over an extended area (1.3 mm x 20 mm) of PLL-coated glass, and have used these patterned substrates in the culture of mouse cerebellar granule neurons. Customized image analysis software is then used to extract relevant neurite growth parameters from statistically meaningful numbers of cultured cells (a few thousand cells per substrate). 5) In collaboration with NICHD, the fabrication and optimization of a device for generating core-shell alginate microcapsules for co-culture of granulosa cells, theca cells, and oocytes, with the goal of understanding oocyte development and maturation. 6) With groups in NIDCD, NCI, NIEHS, and the VRC, the fabrication of microfluidic devices for droplet encapsulation of single cells, with the goal of implementing modified versions of the recently published techniques for massively parallel single-cell DNA sequencing for a variety of applications on campus. 7) The use of a microfluidic channel together with custom instrumentation developed in LCMB, NICHD in order to separate the effects of pressure and rapid (msec) shear transients on cultured neural cells, in order to gain a better understanding of the cellular mechanisms of traumatic brain injury. Our group has fabricated the microchannels, performed modelling aimed at understanding the shear transients experienced by the cells as a function of the bulk flow, and consulted extensively on how best to interface the microchannel with the existing instrumentation. 8) At the instigation of LCIMB, NIBIB, the collaborative design and fabrication of an aluminum-patterned window for use in calibrating the measured settling distances in analytical ultracentrifuges. NIST researchers are currently working on the development and validation of a low-volume, commercially available, certified standard, based on the prototype windows made here. 9) In collaboration with NEI, the development of structures to support long-term retinal culture.